This invention relates to a modified acid dye staining method used for detection of fractions of proteins, etc. separated by electrophoresis, and a reagent solution used therefor.
Fractions of proteins, etc. separated by electrophoresis are usually colorless, and hence their staining with dyes, etc. is always conducted for detection an identification.
For example, amido black 10B (C.I. No. 20470), Coomassie brilliant blue R-250 (C.I. No. 42660) and Acid Violet 17 (C.I. No. 42650) are widely used as acid dyes for staining in dark blue, light blue and light purple, respectively. However, in staining with these acid dyes, the dyes intrude into and settle in a get itself though not bound thereto through stationary bounds such as chemical bonds, so that a colored background appears. Therefore, destaining of the background is indispensable. The destaining is conducted usually by immersion with shaking in a methanol-water mixed solution containing acetic acid or trichloroacetic acid. Although depending on the kinds of the dye used and the gel and other conditions of the staining, the time required for the destaining is at least 4 hours. For making the destaining perfect, the time is usually longer, namely, 6 to 7 hours. The time required for the staining is 2 to 4 hours in practice though a little shorter in some modified methods. Therefore, the total time required exceeds a working day.
On the other hand, Analytical Biochemistry 20, 150 (1967) describes a modified method in which staining is carried out by placing trichloroacetic acid together with Coomassie brilliant blue R-250 immediately before use. Analytical Biochemistry 152, 308 (1986) describes a modified method in which staining is carried out by adding picric acid to the above dye. In the former method, the staining can be carried out in 1 hour, but a destaining procedure is indispensable and the sensitivity is low. In the latter method, the staining can be carried out in 30 minutes, but this method has a serious defect in that destaining of the background requires twice as much time as that usually required, owing to hindrance by the yellow color of picric acid. This method involves a problem of the danger of handling picric acid at a starting material for a reagent solution.
Thus, staining with the acid dyes requires a troublesome procedure and a long time, but it is undeniable that the staining is now the most reliable, useful and frequently used method for detection and identification of fractions separated by electrophoresis.